The esophageal squamous epithelium is characterized by the migration of cells from zones of proliferation to terminal differentiation with eventual desquamation. This process is continuously renewed by disrupted in peptic esophagitis, Barrett's esophagus, and dysplasia. The goal of this proposal is to define the molecular mechanisms underlying transcriptional regulation of esophageal gene expression in the transition from proliferation to differentiation. Two interrelated systems will be employed in this proposal to achieve this goal: the Epstein-Barr Virus (EBV) ED-L2 promoter and the keratin (K4) promoter, both of which are uniquely active in the esophageal epithelium. Preliminary studies have demonstrated that the EBV ED-L2 promoter leads to esophageal squamous specific gene expression in transgenic mice. Analysis of this promoter in esophageal epithelial cell lines has led to the identification of a critical cis-regulatory element that interacts with a transcription factor expressed in esophageal squamous epithelial cells, designed keratinocyte specific factor (KSF). In addition, give that protein kinase C is critical in the maintenance of the proliferation-differentiation gradient in the esophageal epithelium, we have shown that phorbol ester, an activation of protein kinase C, is important in activation the ED-L2 promoter. This is mediated through the USF helix-loop-helix factor and the Kruppel like transcription factors. Finally, K4 is critical in the same esophageal epithelial proliferation differentiation gradient based upon preliminary data that targeted disruption of it in mice leads to impaired differentiation, suggesting that the transcriptional regulatory mechanisms are common to the ED-L2 and K4 promoters. The Specific Aims of the proposal are the following: (1) To clone and characterize KSF that trans-activates the ED-L2 promoter; (2) To determine the role of the USF and Kruppel-like transcriptional factors in the mediation of phorbol ester's effects on the ED-L2 promoter; and (3) To define the transcriptional mechanisms governing K4 regulation through KSF, USF and the Kruppel-like factors. Overall, the study of these integrated Specific Aims will lead to a greater understanding of the molecular basis for the commitment to differentiation in the esophageal squamous epithelium.